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Gentek 2 Essay

528 words - 2 pages

Resume:Formålet med QPCR er, at måle antallet af kopier af Lamda DNA og kvantitativt bestemme indholdet.IndledningFormål:Formålet med QPCR er, at måle antallet af kopier af Lamda DNA og kvantitativt bestemme indholdet.Teori:QPCR er en kvantitativ PCR metode, hvorpå det er muligt at følge "in real time" og få svar på mængden af DNA eller RNA.Metoden for QPCR følger de samme princip som ved PCR. Der er dog ved QPCR-metoden tilsat et stof, SYBR GREEN, så det er muligt at måle og følge processen.Princippet i PCR:Der startes med en hotstart for at klargøre enzymet, for at kunne undgå primer dannelse som f.eks primer-dimere og får at undgå at primerne sæter sig uspecifikt. Ligeledes er det en hjælp til, at man ikke behøver at være så hidsig med at opholde det på is. Derefter startes denatureringen ved 95 grader. Her vil hydrogenbindingerne brydes, så vi går fra dobbelt strenget DNA til enkelt strenget. Herefter sænkes temperaturen ((temperaturen til primerne bestemmes ved at beregne antallet af nukleotider vha. ligningen Tm = 2(A+T)+4(G+C), og her sættes temperaturen 2-5 grader under resultatet) så annealingen kan ske. Her sættes primerne på det specifikke område og gør klar til at Taq polymerasen kan bygge på. Temperaturen øges herefter til 72 grader, så polymerasen kan ske. Her bindes taq polymerasen ved primerne og der sættes nye nukleotider på i forlængelse af den, med den eksisterende streng som skabelon. Processen denaturering-annealing-polymerisering gentages i f.eks 30 gange for at få fordoblet vores target DNA i det pågældende template DNA.SYBR GREEN effekt:SYBR GREEN er et fluorescerende farvestof som bindes...

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